One-step labeling of short 3'-End AF (Alexa Fluor®)-Dye modified RNA is now easily achievable via T4 RNA Ligase 1-mediated incorporation of correspondingly labeled pCps (Fig. 1).
AF (Alexa Fluor®)-Dyes are hydrophilic fluorescent dyes with remarkable photostability. Linker-optimized AF (Alexa Fluor®)-Dye labeled pCps possess excellent substrate properties and thus allow efficient preparation of AF (Alexa Fluor®)-Dye modified RNA probes (up to 100 nt) (Fig. 1) (Tab. 1).
Figure 1: 3'-End AF (Alexa Fluor®) RNA labeling can be performed in a one-step procedure.
RNA oligo (43 nt) was 3'-End labeled using T4 RNA Ligase 1 components in combination with pCp-AF488. Reaction conditions: 50 pmol RNA oligo, 1000 pmol pCp-AF488, 1 mM ATP, 40 U T4 RNA Ligase 1, 1x T4 RNA Ligase 1 Reaction Buffer, 1.33 U RNAse inhibitor, 10 % DMSO, 15 % PEG8000. 18 h incubation at 16 °C. AF488-labeled RNA oligo was subsequently purified from crude reaction mix with Oligo Clean & Concentrator Columns (#4060, Zymo) and analyzed by UV/VIS spectroscopy (A260 nm: AF488-labeled & non-labeled RNA population/ A494 nm: AF488-labeled RNA probe only).
| Product | Dye | Substitute for | Excmax/ Emmax [nm]* |
|---|---|---|---|
| pCp-AF488 | AF488 (also known as Alexa Fluor®488) |
Fluorescein / ATTO488 | 494 / 517 |
| pCp-AF555 | AF555 (structural analog to Alexa Fluor®555) |
Cy3 | 555 / 572 |
| pCp-AF594 | AF594 (also known as Alexa Fluor®594) |
Texas Red | 590 / 617 |
| pCp-AF647 | AF647 (structural analog to Alexa Fluor®647) |
Cy5 / ATTO643 | 648 / 671 |
